scholarly journals Neutralization of a Distributed Coulombic Switch Precisely Tunes Reflectin Assembly

2018 ◽  
Author(s):  
Robert Levenson ◽  
Colton Bracken ◽  
Cristian Sharma ◽  
Jerome Santos ◽  
Claire Arata ◽  
...  
Keyword(s):  
Skin Color ◽  
Colloidal System ◽  
Catalytic Phosphorylation ◽  
Ph Titration ◽  
Sequence Composition ◽  
Coulombic Repulsion ◽  
Spatially Distributed ◽  
Assembly Behavior ◽  
A Charge

AbstractReflectin proteins are widely distributed in reflective structures in cephalopods, but only in Loliginid squids are they and the sub-wavelength photonic structures they control dynamically tunable, driving changes in skin color for camouflage and communication. The reflectins are block copolymers with repeated canonical domains interspersed with cationic linkers. Neurotransmitter-activated signal transduction culminates in catalytic phosphorylation of the tunable reflectins’ cationic linkers, with the resulting charge-neutralization overcoming Coulombic repulsion to progressively allow condensation and concommitant assembly to form multimeric spheres of tunable size. Structural transitions of reflectins A1 and A2 were analyzed by dynamic light scattering, transmission electron microscopy, solution small angle x-ray scattering, circular dichroism, atomic force microscopy, and fluorimetry. We analyzed the assembly behavior of phospho-mimetic, deletion, and other mutants in conjunction with pH-titration as an in vitro surrogate of phosphorylation to discover a predictive relationship between the extent of neutralization of the protein’s net charge density and the size of resulting multimeric protein assemblies of narrow polydispersity. Comparison of mutants shows this sensitivity to neutralization resides in the linkers and is spatially distributed along the protein. These results are consistent with the behavior of a charge-stabilized colloidal system, while imaging of large particles, and analysis of sequence composition, suggest that assembly may proceed through a transient liquid-liquid phase separated intermediate. These results offer insights into the basis of reflectin-based tunable biophotonics and open new paths for the design of new reflectin mutants with tunable properties.

Formation and Structure of Casein Micelles in Lactating Mammary Tissue

1970 ◽  
Vol 28 ◽  
pp. 150-151
Author(s):  
Robert J. Carroll ◽  
Marvin P. Thompson ◽  
Harold M. Farrell
Keyword(s):  
Size Distribution ◽  
G Protein ◽  
Milk Proteins ◽  
Mammary Tissue ◽  
Colloidal System ◽  
Casein Micelles ◽  
The Stability

Milk is an unusually stable colloidal system; the stability of this system is due primarily to the formation of micelles by the major milk proteins, the caseins. Numerous models for the structure of casein micelles have been proposed; these models have been formulated on the basis of in vitro studies. Synthetic casein micelles (i.e., those formed by mixing the purified αsl- and k-caseins with Ca2+ in appropriate ratios) are dissimilar to those from freshly-drawn milks in (i) size distribution, (ii) ratio of Ca/P, and (iii) solvation (g. water/g. protein). Evidently, in vivo organization of the caseins into the micellar form occurs in-a manner which is not identical to the in vitro mode of formation.

scholarly journals An in vitro feasibility investigation considering primary human melanocytes for spray- grafting of freshly isolated autologous skin cells for burn treatment and a clinical case report

2019 ◽  
pp. 1-8
Author(s):  
Jörg C. Gerlach ◽  
C. Johnen ◽  
B. Hartmann ◽  
J. Plettig ◽  
K. Bräutigam ◽  
...  
Keyword(s):  
Cell Morphology ◽  
Skin Color ◽  
In Vitro Model ◽  
Primary Cultures ◽  
Cell Suspensions ◽  
Epidermal Keratinocytes ◽  
Cell Counts ◽  
Skin Coloration ◽  
Vitro Model

A skin cell-spray grafting technique that enables the on-site application of freshly isolated autologous single cell suspensions was already applied in many cases on caucasian patients with low skin coloration. Our project hypothesis is that these suspensions contain keratinocytes and vital melanocytes, that are of particular interest for the treatment of patients of darker skin color. To test this, we applied an in vitro model, wherein the feasibility of i) isolating and ii) spraying of freshly isolated autologous melanocyte-keratinocyte cell suspensions was investigated. Primary human epidermal keratinocytes (HEKs) and melanocytes (MCs) were isolated from skin biopsies (n=8). Biochemical parameter, cell counts, cell morphology, growth behavior and immunofluorescence results were compared in two groups using MC cultures and co-cultures of MCs with HEKs. Case information on using the method clinically with one patient is included. The sprayed mixed cell suspensions proliferated in all groups without measurable loss of viability, and MCs exhibited a regular cell morphology in monoculture up to passage 4°. The sprayed MCs and HEKs demonstrated in vitro glucose and lactate metabolism that was comparable to the pipetted controls. In co-culture, well distributed CK14+ HEKs and NKI/beteb+ MCs could be demonstrated, which interacted in the in vitro model. The ratio of HEKs : MCs in our primary cultures were microscopically counted (n=8 each) as mean +/- SD 1,211,000 (+/- 574,343) HEK : 99,625 (+/- 59,025) MC; i.e., a ratio of approx. 12 : 1. Using the isolation method clinically for a patient with dark skin coloration after suffering severe second-degree burns shows a satisfying re-pigmentation of the resulting wound post healing. Freshly isolated spray-on melanocyte/keratinocyte suspensions provide for a considerable amount of viable HEKs and MCs. Using MCs in spray-grafting suspensions could represent a promising approach for treating severe partial-thickness burns and innovative therapy developments that also aim to address cosmetic aspects.

scholarly journals Dominant Negative Dimerization of a Mutant Homeodomain Protein in Axenfeld-Rieger Syndrome

2003 ◽  
Vol 23 (6) ◽  
pp. 1968-1982 ◽  
Author(s):  
Irfan Saadi ◽  
Adisa Kuburas ◽  
Jamison J. Engle ◽  
Andrew F. Russo
Keyword(s):  
Dominant Negative ◽  
Hill Coefficient ◽  
Homeodomain Protein ◽  
Yeast Two Hybrid ◽  
Autosomal Dominant Disorder ◽  
Rieger Syndrome ◽  
A Charge ◽  
Two Hybrid

ABSTRACT Axenfeld-Rieger syndrome is an autosomal-dominant disorder caused by mutations in the PITX2 homeodomain protein. We have studied the mechanism underlying the dominant negative K88E mutation, which occurs at position 50 of the homeodomain. By using yeast two-hybrid and in vitro pulldown assays, we have documented that PITX2a can form homodimers in the absence of DNA. Moreover, the K88E mutant had even stronger dimerization ability, primarily due to interactions involving the C-terminal region. Dimerization allowed cooperative binding of wild-type (WT) PITX2a to DNA containing tandem bicoid sites in a head-to-tail orientation (Hill coefficient, 1.73). In contrast, the WT-K88E heterodimer bound the tandem sites with greatly reduced cooperativity and decreased transactivation activity. To further explore the role of position 50 in PITX2a dimerization, we introduced a charge-conservative mutation of lysine to arginine (K88R). The K88R protein had greatly reduced binding to a TAATCC element and did not specifically bind any other TAATNN motif. Like K88E, K88R formed relatively stronger dimers with WT. As predicted by our model, the K88R protein acted in a dominant negative manner to suppress WT PITX2a activity. These results suggest that the position 50 residue in the PITX2 homeodomain plays an important role in both DNA binding and dimerization activities.

Tropomyosin-paramyosin system and ‘prolonged contraction’ in a molluscan smooth muscle

1964 ◽  
Vol 160 (981) ◽  
pp. 536-542 ◽  
Keyword(s):  
Slow Relaxation ◽  
Retractor Muscle ◽  
Muscle Fibres ◽  
Colloidal System ◽  
Contraction Phase ◽  
Contractile System ◽  
Molluscan Smooth Muscle ◽  
Anterior Byssal Retractor Muscle

Tonic molluscan muscles such as the anterior byssal retractor muscle of Mytilus ( ABRM ) are outstanding in two respects: First they contain, in even larger amounts than actin and myosin, tropomyosin- A (Bailey 1956; Rüegg 1961 a ) which constitutes a system of filaments, the tropomyosin-paramyosin system (cf. Hall, Jakus & Schmitt 1945; Hanson et al . 1957). Secondly, these muscles are able to maintain tension passively for prolonged periods without fatigue (holding function, catch). The capacity for prolonged (tonic) contraction has been related to the extremely slow relaxation of tonically contracted muscle fibres (Lowy & Millman 1963). During slow relaxation the stretch resistance is high and tension is maintained passively (Johnson & Twarog 1960; Jewell 1959; Lowy & Millman 1963). The slow tension decay after cessation of isometric contraction may then be attributed to the slow breaking of contractile actin-myosin linkages which are assumed to be formed in the preceding contraction-phase (Lowy & Millman 1963). Alternatively Johnson (1962) and Rüegg (1961) suggested that the relaxation of the contractile system is slowed down by a special colloidal system, the tropomyosin-paramyosin system, the stiffness of which was assumed to be increased in the catch and decreased after unlocking the catch with certain types of stimulation, e.g. by addition of serotonin. Subsequently it has been shown that the catch (i.e. serotonin sensitive stiffness) may also be produced in apparently resting ABRM when the muscle is immersed in a solution of increased CO 2 tension (Rüegg & Weber 1963). The caught state may then be regarded as a stretch-resistant state. If the catch is based on the tropomyosin-paramyosin system, tropomyosin- A structures ought to have a capacity to resist stretch and they should have this holding capacity in surviving as well as in glycerol extracted, dead muscle fibres. This possibility has been tested by stretch experiments with surviving and extracted ABRM fibre-bundles after inactivation of the actomyosin contractile system with thiourea. The latter has been shown to be an inhibitor of actinmyosin interaction in vitro (Portzehl 1961; Rüegg 1961 b ) and since it penetrates readily into muscle fibres, we have applied it also in the case of surviving ABRM in an attempt to inactivate the actomyosin system in situ (Rüegg, Straub & Twarog 1963).

scholarly journals On the Aqueous Solution Behavior of C-Substituted 3,1,2-Ruthenadicarbadodecaboranes

Inorganics ◽  
2019 ◽  
Vol 7 (7) ◽  
pp. 91 ◽  
Author(s):  
Marta Gozzi ◽  
Benedikt Schwarze ◽  
Peter Coburger ◽  
Evamarie Hey-Hawkins
Keyword(s):  
Aqueous Solution ◽  
Aqueous Solutions ◽  
Self Assembly ◽  
Self Assembling ◽  
Vis Spectroscopy ◽  
Assembly Behavior ◽  
Biological Performance ◽  
Type 3

3,1,2-Ruthenadicarbadodecaborane complexes bearing the [C2B9H11]2− (dicarbollide) ligand are robust scaffolds, with exceptional thermal and chemical stability. Our previous work has shown that these complexes possess promising anti-tumor activities in vitro, and tend to form aggregates (or self-assemblies) in aqueous solutions. Here, we report on the synthesis and characterization of four ruthenium(II) complexes of the type [3-(η6-arene)-1,2-R2-3,1,2-RuC2B9H9], bearing either non-polar (R = Me (2–4)) or polar (R = CO2Me (7)) substituents at the cluster carbon atoms. The behavior in aqueous solution of complexes 2, 7 and the parent unsubstituted [3-(η6-p-cymene)-3,1,2-RuC2B9H11] (8) was investigated via UV-Vis spectroscopy, mass spectrometry and nanoparticle tracking analysis (NTA). All complexes showed spontaneous formation of self-assemblies (108–109 particles mL−1), at low micromolar concentration, with high polydispersity. For perspective applications in medicine, there is thus a strong need for further characterization of the spontaneous self-assembly behavior in aqueous solutions for the class of neutral metallacarboranes, with the ultimate scope of finding the optimal conditions for exploiting this self-assembling behavior for improved biological performance.

Effect of pH on Cl- transport in TAL of Henle's loop

1987 ◽  
Vol 253 (6) ◽  
pp. F1216-F1222 ◽  
Author(s):  
Y. Kondo ◽  
K. Yoshitomi ◽  
M. Imai
Keyword(s):  
Choline Chloride ◽  
Chloride Diffusion ◽  
Ph Titration ◽  
Cl Transport ◽  
Henle's Loop ◽  
Basolateral Side ◽  
Diffusion Voltage ◽  
Bathing Fluid ◽  
Cl Permeability

To further characterize the mechanism of Cl- transport across the hamster thin ascending limb (TAL) of Henle's loop, we examined effects of pH on Cl- permeability as determined by either the choline chloride diffusion voltage or the lumen-to-bath 36Cl flux in the isolated segments perfused in vitro. When pH of the bathing fluid or the perfusate was reduced from 7.4 to 5.8, the Cl(-)-Na+ permeability ratio (PCl/PNa) was reduced from 2.77 +/- 0.21 to 0.48 +/- 0.02 (n = 7, P less than 0.01) or from 2.55 +/- 0.15 to 0.81 +/- 0.11 (n = 6, P less than 0.01), respectively. At 37 degrees C, when the pH of the bathing fluid was reduced from 7.4 to 6.2, the lumen-to-bath flux coefficient for 36Cl (X10(-7) cm2/s) was reduced from 84.8 +/- 7.5 to 20.4 +/- 3.2 (n = 7, P less than 0.01), whereas the value for 22Na was unchanged (27.3 +/- 2.9 vs. 25.3 +/- 2.5, n = 5). From the pH titration curves for PCl/PNa, pKa values for proton binding were 6.31 and 5.78, and Hill's coefficients were 2.1 and 2.3 on the basolateral side and on the luminal side, respectively. Alkalinization had little or no effect on the Cl- permeability. At room temperature, the acid pH did not affect the Cl- permeability. Intracellular acidification with o-nitrophenylacetate also decreased the Cl- permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals In vivo measurement of the frame-based application accuracy of the Neuromate neurosurgical robot

2015 ◽  
Vol 122 (1) ◽  
pp. 191-194 ◽  
Author(s):  
Daniel von Langsdorff ◽  
Philippe Paquis ◽  
Denys Fontaine
Keyword(s):  
Independent System ◽  
In Vivo Measurement ◽  
Stereotactic Frame ◽  
Spatially Distributed ◽  
The Mean ◽  
Neurosurgical Robot ◽  
Better Than ◽  
Deep Brain

OBJECT The application accuracy of the Neuromate neurosurgical robot has been validated in vitro but has not been evaluated in vivo for deep brain stimulation (DBS) electrode implantations. The authors conducted a study to evaluate this application accuracy in routine frame-based DBS procedures, with an independent system of measurement. METHODS The Euclidian distance was measured between the point theoretically targeted by the robot and the point actually reached, based on their respective stereotactic coordinates. The coordinates of the theoretical target were given by the robot's dedicated targeting software. The coordinates of the point actually reached were recalculated using the Stereoplan localizer system. This experiment was performed in vitro, with the frame fixed in the robot space without a patient, for 21 points spatially distributed. The in vivo accuracy was then measured in 30 basal ganglia targets in 17 consecutive patients undergoing DBS for movement disorders. RESULTS The mean in vitro application accuracy was 0.44 ± 0.23 mm. The maximal localization error was 1.0 mm. The mean (± SD) in vivo application accuracy was 0.86 ± 0.32 mm (Δx = 0.37 ± 0.34 mm, Δy = 0.32 ± 0.24 mm, Δz = 0.58 ± 0.31 mm). The maximal error was 1.55 mm. CONCLUSIONS The in vivo application accuracy of the Neuromate neurosurgical robot, measured with a system independent from the robot, in frame-based DBS procedures was better than 1 mm. This accuracy is at least similar to the accuracy of stereotactic frame arms and is compatible with the accuracy required in DBS procedures.

scholarly journals Molecular Mechanisms of Anti-Melanogenic Gedunin Derived from Neem Tree (Azadirachta indica) Using B16F10 Mouse Melanoma Cells and Early-Stage Zebrafish

Plants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 330
Author(s):  
Hwang-Ju Jeon ◽  
Kyeongnam Kim ◽  
Chaeeun Kim ◽  
Myoung-Jin Kim ◽  
Tae-Oh Kim ◽  
...  
Keyword(s):  
Skin Color ◽  
Molecular Mechanisms ◽  
Early Stage ◽  
Melanoma Cells ◽  
Ultraviolet Rays ◽  
Melanin Production ◽  
Zebrafish Model ◽  
Mouse Melanoma

Melanogenesis represents a series of processes that produce melanin, a protective skin pigment (against ultraviolet rays), and determines human skin color. Chemicals reducing melanin production have always been in demand in the cosmetic market because of skincare interests, such as whitening. The main mechanism for inhibiting melanin production is the inhibition of tyrosinase (TYR), a key enzyme for melanogenesis. Here, we evaluated gedunin (Ged), a representative limonoid, for its anti-melanogenesis action. Melanin production in vitro was stimulated by alpha-melanocyte stimulating hormone (α-MSH) in B16F10 mouse melanoma cells. Ged reduced α-MSH-stimulated melanin production, inhibiting TYR activity and protein amount. We confirmed this result in vivo in a zebrafish model for melanogenesis. There was no sign of toxicity and malformation of zebrafish embryos during development in all treated concentrations. Ged reduced the number of produced zebrafish embryo pigment dots and melanin contents of embryos. The highly active concentration of Ged (100 µM) was much lower than the positive control, kojic acid (8 mM). Hence, Ged could be a fascinating candidate for anti-melanogenesis reagents.

scholarly journals A Colloidal Singularity Reveals the Crucial Role of Colloidal Stability for Nanomaterials In-Vitro Toxicity Testing: nZVI-Microalgae Colloidal System as a Case Study

PLoS ONE ◽  
2014 ◽  
Vol 9 (10) ◽  
pp. e109645 ◽  
Author(s):  
Soledad Gonzalo ◽  
Veronica Llaneza ◽  
Gerardo Pulido-Reyes ◽  
Francisca Fernández-Piñas ◽  
Jean Claude Bonzongo ◽  
...  
Keyword(s):  
Crucial Role ◽  
Colloidal Stability ◽  
Toxicity Testing ◽  
In Vitro Toxicity ◽  
Colloidal System

scholarly journals A high throughput bispecific antibody discovery pipeline

2021 ◽  
Author(s):  
Aude I. Segaliny ◽  
Jayapriya Jayaraman ◽  
Xiaoming Chen ◽  
Jonathan Chong ◽  
Ryan Luxon ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
In Vitro Cytotoxicity ◽  
Functional Screening ◽  
Sequence Composition ◽  
Single Cell Pcr ◽  
Screening Efficiency ◽  
And Function

AbstractBispecific antibodies (BsAbs) represent an emerging class of immunotherapy but inefficiency in the current BsAb discovery paradigm has limited their broad clinical availability. Here we report a high throughput, agnostic, single-cell-based BsAb functional screening pipeline, comprising molecular and cell engineering for efficient generation of BsAb library cells, followed by functional interrogation at the single-cell level to identify and sort positive clones and downstream sequence identification with single-cell PCR and sequencing and functionality characterization. Using a CD19xCD3 bispecific T cell engager (BiTE) as a model system, we demonstrate that our single cell platform possesses a high throughput screening efficiency of up to one and half million variant library cells per run and can isolate rare functional clones at low abundance of 0.008%. Using a complex CD19xCD3 BiTE-expressing cell library with approximately 22,300 unique variants comprising combinatorially varied scFvs, connecting linkers and VL/VH orientations, we have identified 98 unique clones including extremely rare ones (∼ 0.001% abundance). We also discovered BiTEs that exhibit novel properties contradictory to conventional wisdom, including harboring rigid scFv connecting peptide linkers yet with in vitro cytotoxicity comparable to that of clinically approved Blinatumomab. Through sequencing analyses on sorted BiTE clones, we discovered multiple design variable preferences for functionality including the CD19VL-VH– CD3VH-VL and CD19VH-VL–CD3VH-VL arrangements being the most favored orientation. Sequence analysis further interrogated the sequence composition of the CDRH3 domain in scFvs and identified amino acid residues conserved for function. We expect our single cell platform to not only significantly increase the development speed of high quality of new BsAb therapeutics for cancer and other disorders, but also enable identifying generalizable design principles for new BsAbs and other immunotherapeutics based on an in-depth understanding of the inter-relationships between sequence, structure, and function.

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